In this proposal, I describe my plans to elucidate the mechanism by which autoproteolysis regulates bacterial toxin function. In my postdoctoral work thus far, I have discovered a unique mechanism of protease activation and delineated a role for a cysteine protease domain (CPD) in autoprocessing a newly recognized family of bacterial toxins. I have identified the first chemical inhibitors of this novel protease family and have begun developing these inhibitors into chemical probes to study protease substrate specificity and function. In the mentored phase of the proposal, I will continue to learn how to synthesize small molecule inhibitors and probes to monitor and perturb CPD activation in the lab of Dr. Matthew Bogyo at Stanford University. This phase will rely on the depth and diversity of research within the Bogyo lab and the excellent research environment at Stanford. I will supplement my institutional environment with a course in surface plasmon resonance and a visit to a future collaborator's lab (Dr. Jimmy Ballard at Oklahoma State University) where I will learn how to culture Clostridium difficile and purify its glucosylating toxins. At Stanford, I will continue to capitalize on the excellent career development resources within the School of Medicine by attending lectures directed at helping postdoctoral fellows transition into academic positions. I will continue to consult with my advisor, Dr. Bogyo, and an informal mentoring committee, consisting of Dr. John Boothroyd and Dr. K. Christopher Garcia at Stanford, to help me successfully navigate the academic job market, apply for funding, and set-up my own research group. Once I acquire an independent position at a research institution, I will apply the probes and inhibitors developed during the mentored phase to study the function of autoproteolysis in regulating toxin function within cells and in animal models of intoxication. In the long-term, I will apply this new method for imaging protease function, along with my background in bacterial genetics and biochemistry, to study bacterial proteases in diverse systems. 6.B. Research Plan Most secreted bacterial toxins are produced as inactive precursors that become proteolytically activated upon encountering a eukaryotic cell. Whereas eukaryotic proteases typically activate these toxins, a select group of toxins are autoproteolytically activated by an internal cysteine protease domain (CPD). The cytotoxic function of Clostridium difficile glucosylating toxins is activated by the CPD, which itself is activated upon binding the eukaryotic-specific small molecule inositol hexakisphosphate (InsP6). Although autoprocessing is essential for toxin activation, how InsP6 activates the CPD, and when and where in the cell the CPD cleaves these toxins, is unknown. This proposal outlines plans to use small molecules to address these important questions regarding CPD-mediated activation of C. difficile glucosylating toxins. Small molecule inhibitors identified in preliminary studies will be developed into probes that allow CPD activation to be visualized in vitro, within host cells, and in animals. These probes will permit the timing and location of toxin activation to be monitored in real-time by covalently tagging activated CPD. Combined with structural methods, the probes will also facilitate analyses of CPD substrate specificity and function, which will in turn inform strategies directed at inhibiting C. difficile glucosylating toxin function. PUBLIC HEALTH RELEVANCE: Clostridium difficile is the leading cause of nosocomial diarrhea worldwide, and the glucosylating toxins TcdA and TcdB are the primary factors responsible for C. difficile-associated disease. Thus, understanding how the CPD regulates C. difficile toxin activation will inform strategies to alleviate the symptoms of this toxin-mediated disease. By targeting virulence rather than bacterial viability, chemical inhibition of the CPD may reduce the recurrence of antibiotic-induced nosocomial diarrhea.